ES2402672T3 - Use of GDF-5 for improvement or maintenance of dermal appearance - Google Patents

Abstract

Cosmetic, non-therapeutic method, for the prevention Cosmetic, non-therapeutic method, for the prevention Cosmetic, non-therapeutic method, for the prevention, deceleration or inversion of aging processes, deceleration or inversion of aging processes, deceleration or inversion of aging processes, selected from the chemical-thermal set, selected from the chemical-thermal set, selected from the set consisting of a chronological aging and an EU consisting of a chronological aging and an EU consisting of a chronological aging and a photoaging, where the composition of the photo aging, where the composition of the moto-aging, where the composition of the extracellular matrix (ECM) of a mammal is extracellular alteratrix (ECM) of a mammal is extracellular alteratrix (ECM) of a mammal is altered by use of one or more related protein (s) (s) ca by use of one or more protein (s) related to the use of one or more protein (s) related to GDF-5 and / or one or more nucleic acid (s) that were GDF-5 and / or one or more nucleic acid (s) queon the GDF-5 and / or one or more nucleic acid (s) encoding said protein (s), in which one procodes ( n) said protein (s), wherein a pro encodes said protein (s), wherein a GDF-5-related protein is any GDF-5-related protein is any protein-related protein GDF-5 is any protein present in nature or created artificially present in nature or created artificially present in nature or created artificially that comprises a cystine knot domain connte that comprises a cystine knot domain connte that comprises a knot domain of cystine with an amino acid identity of at least 6 an amino acid identity of at least 6 an amino acid identity of at least 60% with the cystine knot domain of 102 aa of 0% with the cystine knot domain of 102 aa of 0% with the cystine knot domain of 102 aa of human GDF-5 (amino acids 400-501 of SEQ ID NO: human GDF-5 (amino acids 400-501 of SEQ ID NO: human GDF-5 (amino acids 400-501 of SEQ ID NO: 1). 1). 1).

Description

Use of GDF-5 for improvement or maintenance of dermal appearance

The present invention is directed to new methods and cosmetic uses of compositions containing growth factor and differentiation proteins. Such compositions are specifically designed to optimize the amount of crucial structural elements of mammalian tissues for the preservation and improvement of the appearance of dermal tissue.

The reduction of structural proteins such as a collagen in various tissues is a problem related to age and / or the environment that appears frequently. In the skin, such gradual or sudden changes are typically accompanied by a decrease in elasticity, by an increase in facial wrinkles and by liver spots, by loss of skin luster and by other unwanted alterations. The process is influenced by various factors including genetic, UV irradiation (ultraviolet), xenobiotics, mechanical stress, hormonal changes and metabolic processes (creation of reactive substances such as radicals, sugars and aldehydes). Taken together, all these factors lead to accumulated alterations of the structure, function and appearance of the skin. As a result, the skin ages.

The influence of the environment, especially solar irradiation with UV rays, is of considerable importance for dermal aging. A photo-aged skin appears wrinkled, lax, thick, with irregular pigmentation and brown spots. Histological and ultrastructural studies have revealed that a photodamaged skin is associated with an increased epidermal thickness, a detriment of the fibrillar organization of collagen and elastin, and alterations in the organization of connective tissue. An aging of the skin due to an exposure to UV rays is superimposed on a chronological aging of the skin. Historically, it has been considered that photoaging and chronological aging of the skin are distinct entities. However, recent evidence indicates that a chronologically aged skin irradiated with UV rays shares important molecular characteristics, including altered signal transduction pathways, which favor the expression of a metalloproteinase (MMP), increased degradation and decreased synthesis of structural proteins, and damage to connective tissue. This concordance of molecular mechanisms suggests that UV irradiation accelerates many key aspects of the chronological aging process in human skin. Even in individuals who are not exposed to excessively dangerous environmental factors, the amount of collagen and other scaffolding substances in the skin tends to decrease with age. For example, a child's skin is rich in collagen III that is partially responsible for the softness of young skin. As body growth slows down, the skin content of type III collagen decreases, while type I content increases for another couple of years. Type I collagen continues to form until about the age of 35, when the skin reaches the peak (maximum) of its mechanical strength. At the age of 60, all types of structural proteins, such as collagen, elastin, fibrillins and decorin, are significantly below their youth levels. Therefore, it is commonly accepted that a notable reduction in the level of protein fibers in the dermis is the main causative agent of skin aging.

The use of natural or synthetic cosmetics to treat the appearance of the face and the condition of the skin is common among many cultures. In the past, various methods for dermal renewal have been developed, but they all have disadvantages. Cosmetics supplemented with a collagen and mucopolysaccharides such as hyaluronic acid to impart moisture to the skin have been conventionally explored to prevent skin aging. Unfortunately, they still could not achieve a sufficient effect for that purpose. Since collagen and other structural proteins are not substantially absorbed if applied extradermally to the skin, these compositions could not increase the activity of fibroblasts and keratinocytes. Retinoic acid as an additive reshapes the skin slowly but at the cost of irritation and chronic redness. Melatonin and vitamin C both increase the skin's collagen but the skin also needs to increase its reconstruction of elastin and water-retaining proteoglycans. Controlled skin damage (eg peels or peels, dermabrasion, lasers) works well only if there is a vigorous regenerative response after therapy by the damaged skin.

In order to prevent the skin from aging and / or to maintain / promote health, it is therefore desired to explore agents that are capable of increasing the production of collagen and other major constituents for the dermis, tissues and tissues. organs The most recent developments of cosmetic compositions (for example described in US Patent Application Document US2002081324) use select growth factor proteins such as platelet-derived growth factor (PDGF), the growth factor epidermal (EGF), and insulin-like growth factors (IGF-I and IGF-II), which have been effective in stimulating the renewal of skin cells. Products containing the recombinant Nfurfuryl-adenine (cinetin) plant growth factor, the epidermal growth factor (ReVive Skincare) and the recombinant TGFβ (CRS, Topix Pharmaceuticals, NY, USA) are already being marketed.

Unlike the compositions described above for the aforementioned purposes, the cosmetic methods and uses of the present invention refer to proteins that are identical to, or are closely related to, the growth / differentiation factor -5 (GDF- 5).

GDF-5 (Hötten et al., 1994., Biochem. Biophys Res. Commun. 204, 646-652) is a morphogen agent that has already been shown to favor cell proliferation, cell differentiation and / or formation of tissue in various tissues. The protein is also known as the morphogenetic protein MP52, the bone morphogenetic protein BMP-14 or the morphogenetic protein derived from cartilage (CDMP-1). The GDF-5 is closely related to the GDF-6 and the GDF-7. These three proteins form a clear and distinct subset of the TGF-β superfamily, thus presenting comparable biological properties and an extraordinarily high degree of identity between amino acid sequences (see, for example, the quote by Wolfman et al. 1997, J. Clin. Invest. 100, 321-330). It has been repeatedly demonstrated that the members of the subset of GDF-5 / -6 / -7 are mainly important inducers and regulators of bone and cartilage (Cheng et al. 2003, J. Bone & Joint Surg. 85A, 1544-1552 ; Settle et al 2003, Developm. Biol. 254, 116-130) as well as the tendon / ligament (Wolfman et al. 1997, J .. Clin. Invest 100, 321-330). All members of this family are initially synthesized as larger precursor proteins, which subsequently undergo proteolytic dissociation together with a cluster of basic residues of approximately 110-140 amino acids from the terminal end of C, releasing the terminal mature protein from C thus some parts of the terminal N domain. Mature polypeptides are structurally related and contain a conserved bioactive domain comprising six or seven canonical cysteine residues, which is responsible for the characteristic three-dimensional "cystine knot" motif of these proteins The native proteins related to GDF-5 are homodimeric molecules and act primarily through interaction with specific receptor complexes that are composed of serine / threonine receptor kinases type I and type II. Receptor kinases subsequently activate Smad proteins and then propagate the signals within the nucleus to regulate the expression of target genes.

Although there have been moderate advances in the specialty, an effective treatment of a decrease related to the age and / or the environment of structural cellular components in the skin remains a great challenge. Therefore, an object of the present invention is to provide new cosmetic methods and uses that have as their goal the preservation and improvement of the appearance of dermal tissue.

Definitions:

In order to avoid misunderstandings and ambiguities, some frequently used terms are defined and exemplified as follows:

The term "cystine knot domain" as used in the present context means the well-known and conserved cysteine-rich amino acid region that is present in the mature protein parts of the TGF-beta superfamily, such as by example the human GDF-5 and forms a three-dimensional protein structure known as cystine knot. In this domain, the respective location of cysteine residues relative to each other is important and is only allowed to vary slightly in order not to lose biological activity. It has been shown that the cystine knot domain alone is sufficient for the biological function of the protein (Schreuder et al. (2005), Biochem Biophys Res Commun. 329, 1076-86). Consensus sequences for cystine node domains are well known in the state of the art. According to the definition given here, the cystine knot domain of a protein begins with the first cysteine residue that participates in the cystine knot of the respective protein and ends with the residue that follows the last cysteine that participates in the cystine node of the respective protein. For example, the cystine knot domain of the human GDF-5 precursor protein (SEQ ID NO: 1) is composed of amino acids 400-501 (see also FIG. 1).

The term "GDF-5-related protein" as used in the present context, means any naturally occurring or artificially created protein that comprises a cystine knot domain with an amino acid identity of at least 60%. with the 102 aa cystine knot domain (amino acids) of GDF-5, human (amino acids 400-501 of SEQ ID NO: 1). This term includes proteins that belong to the set of GDF-5, GDF-6 and GDF-7 proteins from vertebrate or mammalian species, as well as recombinant variants thereof, as long as these proteins show the percentage of Identity mentioned above with the cystine knot domain of human GDF-5. The 60% limiting value is well suited to separate members of the GDF-5 / -6 / -7 protein set as well as certain variants thereof with respect to other additional proteins such as other GDF’s and BMP’s. A comparison of the 102 aa cystine node domain of human GDF-5, human GDF-6 and human GDF-7 (see FIG. 2) reveals the high degree of amino acid identity among these proteins. Human GDF-6 shares 87 (85%), and human GDF-7 shares 83 (81%), identical residues with the GDF-5 cystine knot domain. The respective domains of GDF-5 / -6 / -7 molecules from other vertebrate and mammalian species that have been identified so far, also show very high identity percentages of at least 75% (between 79% and 99 %), when compared to human GDF-5. In contrast, GDF’s and BMP’s that do not belong to

subset of GDF-5 / -6 / -7 have much lower identity values, below 60% (see FIG. 3).

The determination of corresponding amino acid positions in related amino acid sequences, as well as the calculation of the percentages of identity between them, can be easily performed with the help of well-known alignment algorithms and optionally of computer programs using these algorithms. For example, the amino acid identities in this patent application (eg FIG. 2) have been calculated by sequence alignment with the freeware (ClustalX freeware) (version 1.81) with defect parameters and subsequent hand count of identical waste. The defect settings for the pairwise alignment (exact slow) are: for the interstitium opening parameter: 10.00; for the interstitium extension parameter 0.10; for the protein weight matrix: Gonnet 250. The ClustalX program is described in detail in the citation of Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F. and Higgins, D.G. (1997): the ClustalX window interface: flexible strategies for multiple sequence alignment, aided by quality analysis tools. Nucleic Acids Research 24: 4876-4882. ClustalX is a window interface for the ClustalW multiple sequence alignment program and is available for example from various sources, for example by an anonymous ftp from ftp-igbmc.u-strasbg.fr, ftp.emblheidelberg. from, ftp.ebi.ac.uk or through a download of the following web page: http: //www-igbmc.u-strasbg.frlBiolnfo/. The ClustalW program and algorithm are also described in detail in the citation of Thompson, J.D., Higgins, D.G. and Gibson, T.J. 11994): CLUSTALW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice [improving the sensitivity of progressive alignment of multiple sequences by sequence weighting, specific interstitial penalties for certain positions and choice of the weight matrix]. Nucleic Acids Research 22: 4673-4680.

The term "cosmetic composition" refers to non-medical compositions or formulations that are primarily designed to improve the composition and / or function of the skin through the supply of nutrients and / or other bioactive substances. The main objective of cosmetic compositions is to relieve dermal aging and achieve a more attractive appearance of the skin. However, the term "cosmetic compositions" comprises "cosmeceutical" compositions that are sold as cosmetics but nonetheless include comparatively small amounts of pharmaceutically active substances (see Elsner, P., Maibach, H., editing coordinators Cosmeceuticals and active cosmetics (2005) Boca Raton (FL): Taylor and Francis Group.

The terms "dermal", "dermal cells" and "dermal tissues" refer to cells and tissues that are present in the skin of a mammal.

The term "colloid" refers to small particles dispersed in a liquid.

The terms "lipid microparticle formulation" and "LMP formulation" refer to homogeneous microemulsions of the oil / water type that contain small spherical particles having a particle size below 200 nm.

The term "increased solubility" in the context of the GDF-5 related proteins refers to a stabilization of the GDF-5 related protein in blood and / or aqueous solutions at a pH between 4.0 and 9, 0. This stabilization can be achieved by creating a stable liquid or aqueous emulsion formulation, such as colloidal formulations according to the invention.

The term "physiological pH" as used in the present context means a pH within the range of 4 to 8.5 and more preferably within the range of 5 to 7.5.

The term "biological activity" refers to the activity of certain compounds, including, for example, a GDF-5-related protein as measured by the in vitro alkaline phosphatase (ALP) assay, eg such as described in Example 2 or in the quotation from Takuwa et al. (1989), Am. J. Physiol. 257, E797-E803). Appropriate cell lineages, which can be used in said ALP assay, are eg ATDC-5 or MCHT 1/26 cells.

It has now been surprisingly found that cosmetic methods that use GDF-5-related proteins can be efficiently used to stimulate the synthesis of structural elements of mammalian tissues.

The skin of a mammal is the largest organ in the body and it is always changing, which contains many specialized cells and structures. It is composed of three primary layers: the outer layer or epidermis, which provides waterproofing against water and serves as a barrier to an infection; the dermis, which serves as a location for skin flaps; and the basement membrane, which separates the epidermis from the dermis.

The main type of cell that constitutes the epidermis is the keratinocyte. The epidermal skin layer does not contain blood vessels; It is a natural barrier that is responsible for retaining water in the body and protecting it from harmful chemical agents and pathogens. The epidermis is composed of five sub-layers: "stratum basale", "stratum spinosum", "stratum granulosum", "stratum licidum" and "stratum corneum". In the bottom layer (stratum basale = basal stratum) the cells divide and push the already formed cells into higher layers. The upper layer of the epidermis, which is the stratum corneum (= stratum corneum), is made of flat and dead skin cells that move every two days. The basement membrane is a layer of thin but complex tissue that joins the epidermis and dermis of the skin. This anchoring complex is made up of hemidesmosones, anchoring filaments and anchoring fibrils. It contains several layers such as "lamina lucida", which comprises glycoproteins (eg laminin) and collagens. The dermis is the inner layer of the skin and is composed of loose connective tissue. Although it also contains a certain number of cellular structures, which include blood vessels, nerves, hair follicles, smooth muscle, glands and lymphatic tissue, the dermis is relatively acellular. An important part of its volume is the extracellular space, which is filled by an organized network of interwoven macromolecules, which is called extracellular matrix (ECM).

Major components of the ECM of the dermis and other tissues are proteoglycans (eg decorin), mucopolysaccharides (eg hyaluronic acid) and fibrous proteins such as collagens, elastin and fibrillins. Proteoglycan molecules form a "background substance" similar to a highly hydrated gel, in which fibrous proteins are embedded. Hyaluronic acid blocks moisture and supports the structural integrity of the extracellular matrix. Collagen fibers both reinforce and help organize the matrix. Type I and type III collagens are the most common collagens that are present, for example, in the ECM of dermal tissues. Type IV collagen molecules are assembled to give a mesh that constitutes a major part of the mature basal lamina, a thin mat of specialized extracellular matrix that is located below all the leaves and epithelial cell tubes. The basal lamina is attached to the underlying connective tissue by specialized anchoring fibrils made of type VII collagen. The term "basement membrane" is often used to describe the composite material of the basal lamina and this layer of collagen fibrils. Rubber-like elastic fibers, which are composed of elastin and microfibrillar glycoproteins (eg fibrillins-1 and -2) provide the resilience of the extracellular matrix.

In accordance with the detailed and extensive in vivo experiments described in Examples 1 and 2 of the present application, an artificial increase in the concentration of GDF-5 in mammalian skin tissue has important structural implications in the ECM scaffolding. More accurately, for example, the following important effects can be observed:

-

a sharp increase in prominent extracellular matrix proteins: type I collagen (FIG. 6), collagen

type III (FIG. 7), type IV collagen (FIG. 16), elastin (FIG. 8), fibrillin-2 (FIG. 12) and decorin (FIG. 9);

-

a significant increase in the concentration of basement membrane proteins: collagen from

type IV (FIG. 10), type VII collagen (FIG. 11) and fibrillin-2 (FIG. 12);

-

a manifest intensification of the loricrin epidermal differentiation marker (FIG. 13)

-

an increased hyaluronic acid content (FIG. 25)

-

a global reduction in epidermal thickness (FIG. 14)

-

considerable reinforcement of the basement membrane (FIG. 15). Although the effects mediated by GDF-5 are also intertwined in a synergistic manner, it is worth noting that each observed alteration affects the composition of the ECM individually and initiates a discrete regeneration action on dermal cells. The subject of the invention is defined in the present claims. In one embodiment of the invention, the disclosed non-therapeutic and cosmetic methods that use GDF-5 related proteins can be used for the prevention, deceleration or inversion of dermal aging processes, selected from chronological aging and photoaging. . The increase in structural proteins, mediated by growth factors, results in the reversal of a multitude of degenerative effects. Especially preferred is the improvement or prevention of wrinkles, the retardation of dermal aging, the thickening of a basement membrane and / or the reduction of epidermal thickness.

For a better understanding of the scientific context and the impact of the invention, some relevant bibliography that collects the alterations related to age and photodamage (caused by light) in dermal cells is briefly described hereafter.

The skin aging process is related to intrinsic / chronological aging and photo aging. Both types of aging, damage caused to human skin due to repeated exposure to ultraviolet (UV) radiation from the sun (photoaging) and damage that appears due to the passage of time (chronological aging), result in a number of changes in the structure, mechanical properties and function of the skin, ultimately leading to the appearance of wrinkles and a significant reduction in skin elasticity. Mainly among the intracellular alterations caused by chronological aging and photoaging are considerably lower levels of collagen, decorin and other components of the connective tissue of the skin. Studies have shown, for example, that

-

Type I collagen is reduced by 58% in a skin irradiated with UV rays due to an increased production of matrix metalloproteinases that degrade collagen and a decrease in the synthesis of collagen (Fisher et al. 1997: Pathophysiology of premature skin aging induced by ultraviolet light [Pathophysiology of premature aging of the skin induced by ultraviolet light]. N Engl J Med 337: 1419-1428). A significant reduction of type I collagen is also typical of chronological aging, as demonstrated by a comparison of dermal fibroblasts isolated from the skin of young individuals (18 to 29 years old) versus elderly individuals (80 years old). years and more) (Varani and

10 collaborators 2006: Am J Pathol. 168, 1861-1868). - It has been found in anti-aging studies that a significant increase in type III collagen is linked to a marked improvement in skin elasticity and a decrease in wrinkle depth (Schmidt et al. 1996: Treatment of skin aging with topical estrogens [Skin aging treatment with topical estrogens]. Int J Dermatol. 35: 669-674).

15 -a wrinkle-bearing skin, irradiated with ultraviolet B rays, is characterized by an increase in the degradation activity of type IV collagen (Inomata et al. 2003: Possible involvement of gelatinases in basement membrane damage and wrinkle formation in chronically ultraviolet B -exposed hairless mouse [Possible involvement of gelatinases in the damage to the basement membrane and the formation of wrinkles in a shaved mouse, chronically exposed to ultraviolet B rays] J Invest

20 Dermatol. 120,128-34). -the basal expression of the type VII collagen gene decreases in an age-dependent manner in fibroblasts (Chen et al 1994: Type VII collagen gene expression by human skin fibroblasts and keratinocytes in culture: influence of donor age and cytokine responses [ The expression of the type VII collagen gene by fibroblasts and keratinocytes of human skin in culture: influence of the age of the

25 donor and responses to cytokines]. J Invest Dermatol. 102, 205-209). -a chronic irradiation with UVB rays induces a loss of hyaluronic acid from the dermis, thus contributing to the still and mute phenotype of dermal fibroblasts (Dai et al. 2007: Chronic ultraviolet B irradiation causes loss of hyaluronic acid from mouse dermis because of downregulation of hyaluronic acid synthases [Chronic irradiation with ultraviolet B rays causes loss of

30 hyaluronic acid from the dermis of a mouse because of the downward regulation of hyaluronic acid synthases]. Am J Pathol. 171, 1451-1461). -Hyaluronic acid content of human skin decreases in older individuals (Poulsen et al. 1982: Determination of hyaluronic acid, dermatan sulphate, heparan sulphate and chondroitin 4/6 sulphate in human dermis, and a material of reference [Acid determination hyaluronic sulfate

35 dermatan, heparan sulfate and chondroitin sulfate 4/6 in a human dermis, and a reference material]. Scand J Clin Lab Invest. 42, 545-549). - Elastin degeneration is a prominent histological marker of chronological skin aging (Mestre-Deharo et al. 1991: Histologic signs of cutaneous aging. Rev Fr Gynecol Obstet. 86, 425-432).

40-Keratinocytes synthesize fibrillin-2 and assemble foamed microfibrils concurrently with a collagen membrane expression of the basement membrane. These observations suggest that keratinocytes coordinate the assembly of these different structural elements and have important implications for skin reshaping (Haynes et al. 1997: Keratinocytes express fibrillin and assemble microfibrils: implications for dermal matrix organization [Keratinocytes express fibrillin and

45 assemble microfibrils: implications for the organization of the dermal matrix]. Br J Dermatol. 137.17

2. 3.

-

An immunological staining revealed that decorin is greatly decreased within a photodamaged dermal area (Bernstein et al 1995: Differential expression of the versican and decorin genes in photoaged and sunprotected skin. Comparison by immunohistochemical and northern analyses [La

50 differential expression of the versican and decorin genes in a photo-aged skin protected from the sun. A comparison by immunohistochemical and Northern analyzes]. Lab Invest. 72: 662-669). -a comparison between the skin of a set of older volunteers and another of younger volunteers (Sauermann et al. 2002: Age related changes of human skin investigated with histometric measurement by confocal laser scanning microscopy in vivo [Changes related to

55 age of a human skin investigated with histometric measurement by confocal laser scanning microscopy in vivo]. Skin Res Technol 8, 52-56) demonstrates that epidermal thickness increases in skin tissues of older individuals, while the thickness of the basal layer decreases significantly in this set. Therefore, it seems reasonable to develop cosmetic compositions that are capable of stopping or decelerating these age-related thickness changes.

60 As described in the present context, an increased concentration of GDF-5 related proteins in human skin tissues is very useful for generating the desired effects. Transgenic animals with dermal overexpression of GDF-5 have a basement membrane that is 30% thicker than with wild-type animals. The epidermal thickness is significantly reduced (1-2 layers compared to 3-4 layers in the wild type).

65

The results disclosed in this patent application demonstrate that the GDF-5 protein and / or other closely related proteins are of course effective modulators of the ECM composition. The concentrations of substances such as type I collagen, type III collagen, type IV collagen, type VII collagen, fibrillin-2, hyaluronic acid, elastin and decorin, can be significantly altered and increased by application of the methods and of the compositions described herein.

The described structural modifications of the extracellular matrix can be evoked by proteins that occur in nature or are artificially designed, which are closely related to human growth / differentiation factor 5 (hGDF-5). The term "GDF-5 related proteins" includes functionally similar proteins that belong to the set of GDF-5, GDF-6 and GDF-7 vertebrate proteins as well as recombinant variants thereof. Due to an extraordinarily high degree of identity between amino acid sequences (see FIG. 2), this set of proteins exhibits comparable biological properties. A common feature of all GDF-5 related proteins is the appearance of a bioactive cystine knot domain with an amino acid identity of at least 60% with the 102 aa cystine knot domain of GDF-5 human / SEQ ID NO: 1, which is sufficient for the biological function of the protein. As can be seen from FIG. 3, the 60% preferred limiting value separates members of the GDF-5 / -6 / -7 set from the more distantly related GDF’s and BMP’s. Especially preferred proteins have amino acid identities of at least 70%, 80% or 90% with the 102 aa cystine knot domain of human GDF-5.

Non-limiting examples for proteins related to vertebrate and mammalian GDF-5 are precursor proteins and mature proteins of human GDF-5 (disclosed as MP52 in WO95 / 04819 and as human GDF-5 in the quotation of Hötten et al. 1994, Biochem. Biophys Res. Commun. 204, 646-652), GDF-5 / MP52 recombinant human (rh) document (WO96 / 33215), Arg of MP52 (WO97 / 06254); Human MP52's of HMW (WO97 / 04095), CDMP-1 (WO96 / 14335), GDF-5 mouse (Mus musculus) (US Pat. No. 5,801,014), Rabbit GDF-5 (Oryctolagus cuniculus) (Sanyal et al. 2000, Mol Biotechnol. 16, 203-210), GDF-5 chicken (Gallus gallus) (NCBI accession number NP_989669), GDF-5 African nail frog (Xenopus laevis) (accession number to NCBI AAT99303), monomeric GDF-5 (documents WO 01/11041 and WO 99/61611), human GDF-6 / BMP-13 (document US 5,658,882), mouse GDF-6 (No. access to NCBI NP_038554), GDF-6 / CDMP-2 (document WO96 / 14335), human GDF-7 / BMP-12 (document US 5,658,882), mouse GDF-7 (accession number to NCBI AAP97721), GDF-7 / CDMP-3 (WO96 / 143335). Also covered by the invention are GDF-5 related proteins that have additional mutations such as substitutions, additions and deletions, as long as these additional mutations do not completely suppress the biological activity of the proteins. Some preferred variants are mutants of the GDF-5 related proteins that have improved biological activity. For example, one or more residues that are normally present in the human GDF-5 precursor protein (see FIG. 1) are substituted in these mutants by other amino acids: arginine at position 438 of the GDF-5 precursor protein human is replaced by glycine, alanine, valine, leucine, isoleucine, methionine or asparagine; and / or serine 439 is replaced by aspartic acid, glutamic acid, glycine or isoleucine; and / or asparagine 445 is replaced by serine or threonine. In another high activity mutant, methionine 453 and / or methionine 456 are replaced by alanine, valine or isoleucine. Also of particular interest are mutants in which leucine 441 is replaced by proline.

The biological activities of the GDF-5-related proteins can be easily determined with the help of consecrated assay systems. A common in vitro assay known as the alkaline phosphatase (ALP) assay (Takuwa et al. 1989, Am. J. Physiol. 257, E797-E803), which is also described in Example 3, is very useful and preferred. It has been shown that GDF-5-related proteins increase alkaline phosphatase activity, for example in ROB-C26 cells (Yamaguchi et al. 1991, Calcif. Tissue Int. 49, 221-225) as described in WO95104819 , in embryonic ATDC5 cells (Riken Gene Bank, ROB 0565), in mouse stromal cells MCHT-1/26, and in HPDL cells as shown in Nakamura et al. 2003, J. Periodontal Res. 38,597 -605.

For the present cosmetic methods and uses, the GDF-5 related proteins can be applied in both types of cosmetic compositions. Most administration processes require compositions that have an almost physiological pH value. Unfortunately, the closely related GDF-5 and GDF-6 and GDF-7 proteins feature a non-current pattern of surface charge distribution, which is associated with poor solubility at pH values between pH 4 and pH 9. Although GDF-5-related proteins can be administered by means of aqueous solutions that have a pH around or below 4, the most efficient way to solve the problem of solubility is adhesion to a vehicle. of specific colloidal drug with a very small particle size. The interaction between the growth factor and the selected microtextured vehicle efficiently prevents the desired coagulation of the protein at a slightly acidic / basic pH and even at a neutral pH.

In principle, various colloidal vehicles known in the art can be used for cosmetic compositions. Appropriate vehicles are described extensively in the literature (see eg Barrat and

collaborators 2003: Colloidal drug carriers: achievements and perspectives. Cell mol. life sci. 60, 21-37). However, because of the anomalous distribution of loads on the surface of the GDF-5, it is advisable to test and select a colloidal vehicle material that is optimized for the GDF-5 / GDF-6 / GDF-7 family of increase. Among several others, colloidal vehicles presently used are eg liposomes, mixed micelles, microemulsions, lipid microparticles (LMP) and polymeric nanoparticles which are then briefly characterized: Liposomes are very simple structures that are composed of one or more lipid bilayers of amphiphilic lipids, for example phospholipids or cholesterol. The lipophilic remains of the bilayers are turned towards each other and create an internal hydrophobic environment in the membrane. The size of the liposomes varies from 20 nm to a few μm. Mixed micelles are detergent structures that are composed of bile salts, phospholipids, tri, di- and monoglycerides, fatty acids, free cholesterol and fat-soluble micronutrients. Since it is known that long chain phospholipids form bilayers when dispersed in water, the preferred phase of short chain analog compounds is the spherical micellar phase. The interaction between micelles and hydrophobic / lipophilic drugs leads to the formation of mixed micelles (MM). In the human body, they incorporate hydrophobic compounds with low water solubility. Microemulsions are stable and transparent isotropic liquid mixtures of an oil, water and a surfactant, often in combination with a concomitant surfactant. The aqueous phase may contain salt (s) and / or other ingredients and the "oil" may actually be a complex mixture of different hydrocarbons and olefins. In contrast to ordinary emulsions, microemulsions are formed by simply mixing the components and do not require the high shear conditions that are generally used for the formation of ordinary emulsions. The two basic types of microemulsions are the direct (oil dispersed in water, o / w) and the reverse (water dispersed in an oil, w / o). Lipid microparticles can be divided into nanospheres and lipid microspheres. The microspheres are generally defined as small spherical particles made of any material, having a size of approximately 0.2 to 100 μm. Smaller spheres with a size below 200 nm are usually called nanospheres. Lipid microspheres are homogeneous microemulsions of the oil / water type similar to commercially available fat emulsions. They are produced by an intensive sonication process (ultrasonic treatment) or by high pressure emulsification methods (crushing methods). Simple lipid microspheres consisting of microscopic drops of oils in an aqueous dispersion were first investigated for parenteral nutrition (Shenking A. World Rev. Nutr. Diet. 28, 1-111, 1978). Systems based on aqueous emulsions of soybean oil and an outer shell of lecithin (phosphatidylcholine) (Mizushima Y. Drugs Exptl. Res. XI (9), 595-600, 1985) have also been developed. Natural surfactant lecithin decreases the surface tension of the liquid, thus acting as an emulsifying agent (Seki et al., J. Controlled Release 28, 352-353). Polymeric nanoparticles serve as vehicles for a wide variety of ingredients. The active components can be either dissolved in the polymer matrix or trapped or adsorbed on the surfaces of the particles. Suitable polymers for the production of organic nanoparticles include cellulose derivatives and polyesters such as a poly (lactic acid), a poly (glycolic acid) and their copolymers.

The creation and testing of these and other colloidal drug vehicles are a routine matter that can be done without undue assessment. However, the authors of the invention have illustratively created and tested several colloidal drug vehicles in combination with GDF-5 (see Examples 4 and 5). According to the tests, polymeric nanoparticles (eg, poly- (DL) -lactide PLA resomer® R202H, from Boehringer Ingelheim, Ingelheim, Germany) are capable of incorporating medium-sized protein scales related to GDF -5 (up to 66 g / mg). Since cosmetics usually contain lower amounts of active ingredients than pharmaceutical agents, nanoparticles of this class were considered to be suitable carriers for polymeric compositions. Also, microemulsions of the oil-in-water type, comprising GDF-5-related proteins, glycerol and emulsifying agents, have been successfully created and tested (see Example 4). In addition, some formulations of lipid microparticles (LMP’s) were specifically designed. According to the test results, LMP’s can transport high amounts (up to 2.5 mg / ml) of GDF-5 at a physiological pH (pH 7). Therefore, lipid microparticles are appropriate protein vehicles related to GDF-5.

Numerous natural and synthetic lipids and oils are available that can be used as lipid carriers in colloidal formulations such as an LMP formulation. Synthetic lipids and oils are recommended due to their purity and known chemical properties. All kinds of synthetic oils / lipids can be used, as long as they are biocompatible, for example synthetic saturated oils or esters such as ethyl palmitate, isopropyl palmitate, alkyl myristates, such as isopropyl, butyl and cetyl. , hexyl stearate, triglycerides (eg octanoic or decanoic acids, medium chain triglycerides such as Miglyol® 812), cetyl ricinoleate, stearyl octanoate (purcellin oil) and a hydrogenated polyisobutene. Due to their well-known biocompatibility and other characteristics, common vegetable oils such as eg cottonseed, soybean, sesame, sunflower, safflower, safflower, and seed oils are especially suitable in most cases. Olive, avocado, peanut, walnut, almond and hazelnut. The most preferred oils are olive oil, soybean oil and safflower oil, which have been tested in a satisfactory manner. Other natural oils can be similarly used.

Otherwise, the formulation may comprise an emulsifying agent. Phospholipids such as eg phosphatidylserine, phosphatidylcholine or phosphatidylethanolamine are preferred, preferably in a similar total percentage (± 25 percent w / w, w / vov / v) [p = weight, v = volume] as the vehicle of lipids A commercially available and successfully tested emulsifying agent is Phospholipon ®, a fraction of soy phospholipids comprising at least 80% phosphatidylcholine. Other emulsifying agents are also possible, eg distilled monoglycerides, mono- and diglycerides, acetic acid esters of monoglycerides, organic esters of monoglycerides, sorbitan esters of fatty acids, propylene glycol esters of fatty acids, and polyglycerol esters of fatty acids.

The optimal concentration of the oil and / or the emulsifying agent contributes to the effective generation of bioactive lipid microparticles containing GDF-5. By way of illustration, the sets of LMP formulations of GDF-5 containing low (5-15 mg / ml), intermediate (16-30 mg / ml) and high (31-50 mg / ml) concentrations as well as up to 100 mg / ml) of an oil and identical amounts of the phosphatidylserine emulsifying agent in a succinate buffer were tested in the ALP test system. All formulations proved to be bioactive. However, the activity had a maximum in the intermediate set (see FIG. 21). According to these results, LMP formulations may comprise concentrations of an oil and / or emulsifying agents in the range of 5 to 100 mg / ml. Preferred LMP formulations for use according to the invention comprise concentrations of an oil and / or emulsifying agents comprised between 16 and 30 mg / ml. Concentrations between 20 and 25 mg / ml are highly preferred.

The concentrations of GDF-5 related proteins in the compositions intended for use according to the invention should be chosen depending on the mode and the period of application. Basically, GDF-5-related proteins are extremely potent cytokines that are capable of producing effects even in small amounts. As it can be easily determined with the help of different biological test systems such as eg the alkaline phosphatase assay described herein, a concentration of 0.1 pg of GDF-5 per ml of the respective solution is sufficient to cause biological actions (see dose determination according to FIG. 20). Correspondingly, low concentrations are preferred, varying eg from 0.1 pg / ml to 1 ng / ml or less, if the cosmetic compositions of the invention are administered repeatedly, for example in terms of skin creams or Beauty applied daily. However, maximum effects can be achieved with higher concentrations of the growth factor. In the test system shown in FIG. 20, 1 -100 ng / ml of GDF-5 are highly effective. A dose-independent analysis of the action of GDF-5 using a wide range of serial dilutions (0.3-80 ng / ml, Farkas et al. 1997, Neurosci. Lett. 236, 120-122) gave some Optimum results at a concentration of 20 ng of GDF-5 per ml. In vivo skin models (see eg Example 2 / Fig. 16), commonly use high doses of 1-10 μg / ml. Therefore, in a preferred embodiment of the invention, the cosmetic compositions contain GDF-5-related proteins at concentrations between 0.1 pg / ml and 10 μg / ml. Preferred total doses of GDF-5-related proteins in the case of one-time administrations (eg injections) range from 10 ng to 10 μg.

Because of their small size and good biocompatibility, the compositions can generally be administered by a variety of routes that are well known in the art. The cosmetic compositions can preferably be applied topically, subcutaneously or transdermally.

The compositions may also comprise another bioactive protein (s) in addition to the GDF-5 related proteins. It has been shown that a TGF-β increases the size of a regenerated dermis and stabilizes the dermoepithelial junction (Fitzpatrick and Rosen, J. Cosmet. Laser Ther, 5: 25-34 (2003)). A cocktail (TNS Recovery Complex, from SkinMedica, Inc. Carlsbad, CA, USA) containing seven cytokines (VEGF, IL-6 and -8, HGF, PDGF-a, GCSF, and TGF-β1) derived from Neonatal foreskin fibroblasts were tested in a multi-center study. An evaluation showed an improvement in skin texture, and decreased wrinkling (Rokhsar,

C.K. and collaborators., Dermatol. Surg. 31: 1166-1178 (2005)). Recombinant epidermal growth factor (ReVive Skincare); and the plant growth factor N-furfuryl-adenine (cinetin) are also on the market. All these proteins can be used in conjunction with the GDF-5 related proteins. Other proteins that act synergistically if combined with GDF-5-related proteins are disclosed in the literature and in patents, for example, eg in WO 99/15191. Neurotrophins, hedgehog proteins and transformative growth factor family proteins are preferred, including but not limited to TGF-alphas, TGF-betas, activins, BMP’s and GDF’s. A combination with any of the EGF, TGF-β1, TGF-β2, TGF-β3, NGF and / or GDNF is especially preferred.

Other acceptable components in the compositions are:

-

retinoids (derivatives of (vitamin A) that preserve the integrity of mucosal / epithelial surfaces;

-

hydroxy acids (organic carboxylic acids further classified into hydroxy alpha acids (AHA) and hydroxy beta acids (BHA)) that increase epidermal shedding, eg glycolic acid, lactic acid, citric acid, mandelic acid, malic acid and tartaric acid ;

-

 antioxidants that counteract the harmful effects of free radicals, eg vitamin C, vitamin E, panthenol, lipoic acid, ubiquinone, niacinamide, dimethylaminoethanol, spin traps, melatonin, catalase, glutathione, superoxide dismutase, peroxidase, glucopyranosides, polyphenols , cysteine, allantoin, furfuryl adenine, uric acid and carnosine;

-

 depigmenting agents that relieve hyperpigmentation, eg N-acetyl-4-S-cysteinylphenol, kojic acid, arbutin, azaleic acid, a paper blackberry composition, chemical peeling agents (resorcinol, acid salicylic), the Kligman formula, the Pathak formula and the Westerhof formula; Botanical products, eg camomile, ginseng, Gingko biloba, curcumin, glycyrrhizin, capsaicin and aloe vera; -glycosaminoglycans that support epidermal regeneration, eg hyaluronic acid;

-

 anti-cellulite agents that mediate a lipolysis, eg beta-adrenergic stimulating agents such as theobromine, theophylline, aminophylline, caffeine, epinephrine and alpha 1 adrenergic stimulants such as yohimbine, piperoxane and phentolamine;

-

enzymes such as papain and DNA repair enzymes;

-

hormones, eg estrogens, progesterone, testosterone, and a growth hormone;

-

 antimicrobial agents, eg triclosan, chlorhexidine, povidone iodine, hydrogen peroxide, anti-dandruff preparations and zinc pyrithione;

-

chemical UV filters, eg 3-benzylidene camphor (3-BC) or 4-methyl-benzylidene camphor (4-MBC);

-

 otherwise, buffers, stabilizing agents, preservatives, reducing agents, antioxidant chelating agents, agents that modify isotonicity, deodorants, anesthetics, adjuvants and additives that increase solubility.

These are only non-limiting examples of possible additives, and a professional skilled in the art can easily add other excipients that are in use in cosmetic preparations or that are generally considered safe.

It is further preferred that cosmetic compositions comprising GDF-5 related proteins be administered in typical forms for dermal products. Consecrated and standardized formulations that are well known in the art include, for example, formulations of lipid microparticles, microemulsions of the types of water in oil and oil in water, lotions, gels, creams, ointments, projection formulations of sprays, lipsticks, plasters, facial packs, bath agents, facial cleansers, bath detergents, bath soaps, sun creams, sun lotions, self tanning lotions, after sun exposure lotions, emulsions of the type of oil in water, emulsions of the type of water in oil, emulsions w / o / w, emulsions of the type o / w / o and milky lotions.

The compositions can be prepared under N2 (gaseous) using buffers in which N2 (gaseous) is bubbled to remove dissolved oxygen, or by using a vacuum to remove dissolved gases and to protect lipid components and protein related to GDF-5 regarding oxidation. Alternatively, other inert gases such as argon can be used.

As demonstrated in Example 1 in conjunction with FIG. 4-15, another effective method of carrying out the invention is to use a transgenic approach. Since gene therapy strategies require the transfer of a genetic material to target cells, a further issue of the present invention is the use of a nucleic acid encoding a GDF-5 related protein within the context of the present invention. The nucleic acid may be a DNA sequence and / or an RNA sequence, as long as a GDF-5 related protein, as defined herein, is obtainable from this nucleic acid upon expression. Base triplets encoding amino acids and the degeneracy of the genetic code are generally known. In the present Example 1, a transgenic experiment was performed according to a strategy originally published by Greenhalgh et al. (Oncogene 8 (1993), 2145-2157) and Sellheyer et al. (Proc. Natl. Acad. Sci. USA. 90 ( 1993), 5237-5241). In the described example, a specific skin promoter (human keratin promoter 1) is used. Although the use of specific skin promoters is preferred, other promoters may be similarly employed. As a natural thing, other gene transfer strategies and other vectors that are known in the art can also be used. Many of these gene transfer vectors are based on modified RNA and DNA viruses (derived from retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, poxviruses and others) and have been selected as gene delivery vehicles. because of its ability to efficiently deliver foreign genes associated with efficient gene expression. Viral vectors are used in more than 70% of clinical gene therapy tests worldwide. Current and emerging virus-based genetic engineering strategies for the supply of therapeutic molecules are discussed at the appointment of Mancheno-Corvo et al. 2006: Viral gene therapy. Clin Transl Oncol. 8, 858-67. However, non-viral vectors and transfer techniques, eg cationic vehicles, can also be used (see Pietersz et al. 2006: Structure and design of polycationic carriers for gene delivery [Structure and design of polycationic vehicles for gene delivery] Mini Rev Med Chem. 6, 1285-1298), electroporation or liposomes (Miyazaki et al. 2006: Gene transfer using nonviral delivery systems. Perit Dial Int. 26, 633-640) .

The following non-limiting Examples, together with the figures and sequence protocols are intended to further illustrate the invention.

SEQ ID NO: 1 shows the protein sequence of the human GDF-5 precursor.

SEQ ID NO: 2 shows the DNA sequence of the human GDF-5 precursor.

SEQ ID NO: 3 shows the 120 aa protein sequence of a mature human GDF-5. If produced recombinantly, the protein may alternatively consist of 119 aa, thus beginning with the second aa (proline) of SEQ ID NO: 3.

SEQ ID NO: 4 shows the 120 aa protein sequence of a mature human monomeric GDF-5. The protein may alternatively consist of 119 aa beginning in this way with the second aa (proline) of SEQ ID NO: 4.

FIGURES

FIG. 1 shows additional features of the human GDF-5 precursor protein according to SEQ ID NO: 1:

pre-dominance of aa 001-381 (bold letters) signal peptide aa 001-027 (bold and underlined) part of mature protein from aa 382-501 cystine knot domain of aa 400-501 (underlined).

FIG. 2 shows a comparison of the 102 aa cystine node domain of human GDF-5 (SEQ ID NO: 1), human GDF-6 (sequence 2 from US 5,658,882) and human GDF-7 (sequence 26 from US 5,658,882). The amino acid residues that are identical in all three molecules are highlighted in black.

FIG. 3 shows a table with the sequence identities of cystine node domains of several BMP’s and GDF’s known to the human GDF-5 cystine node domain.

FIG. 4 is a schematic diagram of the basic genetic artificial construction of Example 1 that is composed of a 7 kb human keratin promoter cassette 1, containing an open reading frame of human GDF-5 (hMP52).

FIG. 5 is a map of the transgenic vector pHK1-MP52 according to Example 1.

FIG. 6 shows representative immunofluorescence images that visually reproduce the amount and distribution of type I collagen in the NDE of wild-type mice and that overexpress GDF-5 according to Example 1. Samples were taken from the skin dorsal of mice with an age of 6 months. Collagen I is packed more densely in the extracellular matrix of transgenic animals. A semi-quantitative evaluation of staining intensity supports a significant overall increase (approx. 10-20%) of the deposition of collagen I.

FIG. 7 shows representative immunofluorescence images that visually reproduce the amount and distribution of type III collagen in the NDE of wild-type mice and that overexpress GDF-5 according to Example 1. Samples were taken from the dorsal skin of mice with an age of 5 months. Collagen III is packed more densely in the extracellular matrix of transgenic animals. A semi-quantitative evaluation of staining intensity supports a significant overall increase (approx. 8 -15%) of collagen I deposition.

FIG. 8 shows representative immunofluorescence images that visually reproduce the amount and distribution of elastin in the ECM according to Example 1. Samples of the dorsal skin were taken from mice with an age of 11 months. Significantly more amount of elastin is deposited in skin tissues of transgenic animals.

FIG. 9 shows representative immunofluorescence images that visually reproduce the amount and distribution of decorin in the ECM of wild-type mice and that overexpress GDF-5 according to Example 1. Decorin is a structural element associated with the collagen of the extracellular matrix. Samples were taken from the ventral skin of mice with an age of 11 months. The evaluation of staining intensity indicates that a considerably higher amount of decorin is present in the skin of transgenic animals.

FIG. 10 shows representative immunofluorescence images that visually reproduce the amount of type IV collagen in the basement membranes of wild-type mice and that overexpress GDF-5 according to Example 1. Dorsal skin samples were taken. of mice with an age of 11 months. The results demonstrate a noticeable thickening of the basement membranes in skin samples of transgenic animals. Collagen IV deposition is manifestly increased in basement membranes of transgenic mice.

FIG. 11 shows representative immunofluorescence images that visually reproduce the amount of type VII collagen in the basement membranes of wild-type mice and that overexpress GDF-5 according to Example 1. Samples of the dorsal skin of mice were taken With an age of 11 months. The skin of transgenic animals comprises thicker basement membranes with high concentrations of collagen VII.

FIG. 12 shows representative immunofluorescence images that visually reproduce the amount and distribution of fibrillin-2 in skin samples from wild-type mice and that overexpress GDF-5 according to Example 1. Dorsal skin samples were taken from mice with an age of 11 months. Fibrillin2 is predominantly located in basement membranes and is also a component of the NDE. Transgenic animal membranes are characterized by significantly higher concentrations of fibrillin-2, especially in basement membranes.

FIG. 13 shows representative immunofluorescence images that visually reproduce the amount of loricrin in skin samples from wild-type mice and that overexpress GDF-5 according to Example 1. Samples of dorsal skin were taken from mice with an age of 11 months Loricrin is an important marker of late epidermal differentiation. As indicated by a stronger fluorescence signal in the epidermis of transgenic animals, GDF-5 acts manifestly as a differentiation factor in dermal tissues with profound effects on structural proteins and other cell components.

FIG. 14 shows representative electron microscopy images documenting the dermal thickness changes induced by GDF-5 in transgenic mice according to Example 1. Samples of the dorsal skin were taken from mice aged 11 months. While three to four layers of epidermal cells are present in wild-type animals, dermal samples from mice that overexpress GDF-5 comprise only 1-2 epidermal layers.

FIG. 15 visually reproduces representative electron microscopy images showing the thickening induced by GDF-5 of basement membranes in skin samples of transgenic animals according to Example 1. Samples of the dorsal skin of mice with an age of 11 were taken months The results indicate that the basement membranes of mice that overexpress GDF-5 (with "lamina lucida" [ll] and "dense lamina" [ld]) are thicker by approximately 30% than in wild-type animals.

FIG. 16 shows representative immunofluorescence images documenting the increase induced by GDF-5 in a dose-dependent manner of the type IV collagen in the skin of mice according to Example

2. The upper left image shows a mouse with a transplanted GDF-5 containing a gel vehicle (covered with a silicone hat) according to Example 2. Important dose-dependent epithelial alterations were observed at three weeks later of the administration of 1 or 10 μg of GDF-5. A neovascularization as well as an increased formation of various structural cellular components (eg image: collagen IV) were visually reproduced by classical immunofluorescence techniques.

FIG. 17 shows the general synthesis scheme of polymeric nanoparticles (by the double emulsion method) described in Example 4.

FIG. 18 shows an image taken on a Resomer® R202H polymer nanoparticle reflection electron (REM) microscope according to Example 4.

FIG. 19 demonstrates the effect of an ultrasonic treatment that is mandatory in the production of lipid microparticle (LMP) compositions according to Example 4. Some samples (top image) containing 22.93 μl of olive oil (21, 1 mg), an equal weight of phosphatidylserine and 956 μl of a 50 mM succinate buffer (pH 4.25) were treated with ultrasound for 1 hour. After the ultrasound treatment, the previously milky emulsion (upper image) became transparent with an amber color when held in front of the light (lower image), indicating that the emulsion spheres were becoming smaller and reached the expected diameter of approximately 50 nm.

FIG. 20 shows the results of an in vitro dose determination study of GDF-5 (promotion of cell survival). GDF-5 is effective over the entire range of concentrations tested (from 0.1 pg / ml to 100 ng / ml).

FIG. 21 shows a comparison of the biological activities of three sets of GDF-5 lipid microparticle formulations comprising different concentrations of phosphatidylserine and olive oil (low / set 1: 5-15 mg / ml, represented in Figure 3 8.44 mg / ml, intermediate / set 2: 16-30 mg / ml, represented by 21.1 mg / ml, high / set 3: 31-50 mg / ml; represented by 42.2 mg / ml) . The assemblies were tested in the ALP test system according to Example 3. OD = optical density.

FIG. 22 shows the results of protein size separation by means of an SDS-PAGE according to Example 5. LMP granules with the encapsulated GDF-5 were heated for 10 min at 80 ° C to solubilize lipids. Only very few degradation products are visible, thus demonstrating the stability of the GDF-5 protein. The clues from the left to the right are: 1: marker protein, 3-5: reference standard of GDF-5 5 μg, 0.25 μg and 0.025 μg, 7-9: LMP samples of GDF-5 (5 μg each).

FIG. 23 shows a general scheme of a penetration cell (cell) for the determination of dermal absorption and percutaneous penetration of cosmetic compositions according to Example 5.

FIG. 24 demonstrates the overall structure and organization of a human skin.

FIG. 25 shows representative immunofluorescence images that document the high content of hyaluronic acid (red spot, eg arrow) as a substance that fixes water in the extracellular matrix of animals that overexpress GDF-5 according to Example 1. they took samples of the dorsal skin of mice with an age of 11 months. Compared to wild-type mice, a quantitative evaluation of staining intensity indicates a significantly higher concentration (approx. 20-30%) of hyaluronic acid in the skin of transgenic mice, thereby considerably increasing the moisture content of the skin.

FIG. 26 shows an elastin stain according to van Gieson of skin samples from transgenic wild-type animals that overexpress GDF-5 according to Example 1 (see eg Garvey, Winsome et al. (1991): (a stain of modified Van Gieson Elastic of Verhoff J. Histotech. 14, (2), 113-114) Samples of the dorsal skin of mice with an age of 11 months were taken. High numbers of elastic fibers are predominantly present in the transgenic samples, which appear as bluish black rods (see arrows).

EXAMPLES

Example 1: Skin structure alterations in vivo

This set of experiments, which use an in vivo model of a transgenic mouse, was designed to determine whether an increased intracellular concentration of GDF-5-related proteins is useful for increasing the content of crucial structural components of the ECM such as e.g. Collagens, fibrillins, elastin and decorin. The concentrations of these scaffolding substances decrease during life, for example as a result of aging, photodamage or various disorders.

Following the strategy used by Greenhalgh et al. (Oncogene 8 (1993), 2145-2157) and Sellheyer et al. (Proc. Natl. Acad. Sci. USA. 90 (1993), 5237-5241), genetic constructions were created for the overexpression of human GDF-5 (MP52) in the proliferative basal compartment of the epidermis. The basic artificial construction (FIG. 4) is composed of an open reading frame (ORF) of human GDF-5 (hMP52) that was placed in a directed manner within a 7 kb human keratin 1 (HK1) promoter cassette using artificially created BamHl and Clal restriction sites. Subsequently, an EcoRI fragment of the HKI promoter comprising the GDF-5 ORF (hMP52) was cloned into a plasmid vector and resulted in the transgenic vector pHK1-MP52 (FIG. 5).

An artificial transgenic HK1-GDF-5 construct was amplified and isolated from plasmid pHK1-MP52 by digestion with Hpal and AatII. The 8.2 kb fragment was then purified, filtered using a micro-centrifugation column (Ultrafree-MC, from Millipore) and diluted to a concentration of 5 ng / μl in a microinjection buffer. Female mice with an age of 3 weeks (CB6F1 / Charles River Laboratories breed) were superovulated and mated with male C57BL6 mice (from Charles River Laboratories). The resulting fertilized CB6F2 oocytes were collected from the oviduct of plugged female mice, and cultured until two clear clear pronuclei were visible. The artificial transgenic construction was microinjected into the male pronuclei and injected embryos were grown overnight to reach the stage of two cells. 263 embryos in the two-cell stage were then implanted into the oviduct of pseudo-pregnant foster mothers at 0.5 days after intercourse. In total, three married transgenic animals were identified. The results of the DNA microinjection revealed that 3 of 40 (7.5%) animals had integrated transgenic DNA, a fact that is compatible with the statistical success rate of 5-25% of this technology. Each of the married transgenic animals were crossed with wild-type mice

testing them for the transmission of the germ lineage. Among the F1 generation, transgenic mice could be identified by PCR (polymerase chain reaction). Transgenic mice show a reduced lining with hairs and significant changes in the structure of the skin but no major behavioral abnormalities (FIG. 6-15). Cryogenic sections of dorsal or ventral skin of F1 animals with an age of 5 and 11 months were examined by classical immunofluorescence and electron microscopy techniques.

Cryogenic skin sections revealed a sharp increase in prominent extracellular matrix components such as type I collagen (FIG. 6), type III collagen (FIG. 7), elastin (FIG. 8 and FIG. 26), fibrillin2 (FIG. 12) and decorin (FIG. 9), a clear and distinct increase in the concentration of basement membrane proteins, type IV collagen (FIG. 10), type VII collagen (FIG. 11) and fibrillin -2 (FIG. 12), an intensification of the loricrin epidermal differentiation marker (FIG. 13), a global reduction of epidermal thickness (FIG. 14), a considerable strengthening of the basement membrane (FIG. 15) and a content increased hyaluronic acid (FIG. 25).

Example 2: Transplant assay of in vivo matrix inserted surfaces

In order to study the effects of GDF-5 protein administered directly to dermal tissues, a matrix-inserted surface transplant assay was used. Briefly said, gel supports were loaded with different amounts (in 10 animals each time with 1 μg / ml or 10 μg / ml) of recombinant human GDF-5. A circular area of skin was removed from the back of mice devoid of immunity and covered with gauze-filled gauze. Subsequently, gel holders containing GDF-5 were mounted between 2 rings, covered with a silicone hat and transplanted onto muscular fascia of the back (FIG. 16). At 1 to 4 weeks after transplantation, samples of the dermis were taken and examined by cytohistochemical techniques.

Important dose-dependent epithelial alterations were observed at 3 weeks after application of 1 or 10 μg of GDF-5 (FIG. 16). Neovascularization as well as an increased formation of structural cellular components (collagen I, IV, VII, fibrillins, elastin, decorin, hyaluronic acid) were visually reproduced by classical immunofluorescence techniques. In summary, direct administration of GDF-5 to skin tissues evoked comparable effects such as overexpression of GDF-5 in a transgenic mouse skin (Example 1).

Example 3: Alkaline phosphatase (ALP) assay of a biological activity

The biological activity of GDF-5 related proteins and their colloidal formulations can be easily determined with the aid of consecrated assay systems. The common alkaline phosphatase (ALP) assay (Takuwa et al 1989, Am. J. Physiol. 257, E797-E803) is extremely useful and preferred. In this in vitro assay system, the biological activity of growth factors related to GDF-5 is measured after concomitant culture of different protein concentrations with osteogenic / chondrogenic cells. GDF-5 and proteins related to an osteo- / chondrogenic potential increase the expression of alkaline phosphatase (ALP) in these cells, eg in ATDC-5, ROB-C26 or MCHT-1/26 cells. The activity of ALP in these cell lysate materials is determined by a colorimetric assay. The reaction is based on the hydrolysis of p-nitrophenyl phosphate (PNPP) to give p-nitro-phenol, which becomes visible under alkaline conditions such as the yellow p-nitro-phenol anion. The goal was to measure the activity of the LMP formulations tested by comparison with the activity of ALP obtained with known concentrations of the GDF-5 reference standard.

In a normalized ALP assay, 1x104 cells of ATDC-5 or MCHT1 / 26 cells were incubated overnight in 96-well plates in a cell culture medium (alpha-MEM, penicillin / streptomycin, 2 mM Lglutamine, FCS (10% fetal calf serum) at 37 ° C, 5% CO2, saturated with H2O. The next day, the cells were stimulated with the GDF-5-related proteins or formulations thereof for 72 hours with indicated concentrations of ligands. Subsequently, the cells were washed with a PBS (phosphate buffered saline). A cell lysis was performed in 100 µl of an alkaline lysis buffer 1 (0.1 M glycine, pH 9.6, 1 NP-40, 1 mM MgCl 2, 1 mM ZnCl 2) for 1 h at room temperature. Then 100 µl of alkaline lysis buffer 2 (0.1 M glycine, pH 9.6, 1 NPM-40, 1 mM MgCl2, 1 mM ZnCl2 + 2 mg / ml PNPP) was added. The plates were incubated at 37 ° C, 5% CO2, saturated with H2O. The ALP reaction was thereafter stopped with 100 µl of 30 g / l NaOH and finally the optical density was measured with an automatic microplate reader at 405 nm taking into consideration the subtraction of the empty value.

Example 4: Development of colloidal formulations of basic character of GDF-5 for use in cosmetic compositions

Appropriate GDF-5 formulations can be prepared using different colloidal drug vehicles. Basic compositions comprising colloidal vehicles can comprise eg polymeric nanoparticles and lipid microparticles that can be prepared as follows:

A. Polymeric nanoparticles Multiple polymeric nanoparticles were tested as colloidal drug carriers for GDF-5, eg resomers® (from Boehringer Ingelheim, Germany) with different compositions and molecular weights: the resomers® of PLA R202H and R202S (poly - (DL) -lactide), the resomers® of PLGA RG502, RG502H, RG503H and RG504H (poly- (DLlactide-co-glycolide)). As a general acidic solvent for GDF-5, 10 mM HCl was used, the emulsifying agents investigated were a polyvinyl alcohol (PVA), poloxamer188 (P188) and a poly (vinyl pyrrolidone) (PVP). The synthesis of nanoparticles was carried out with the double emulsion method under sterile conditions according to FIG. 17 (eg phase w1 = 500 μg of protein in 142.1 μl of 10 mM HCl, phase w2 = 160 mg of an emulsifying agent in 30 ml of water, phase o = 40 mg of a polymer in 3.1 ml of methylene chloride). Emulsions were created by ultrasonic treatment for 40 s (seconds) (w1 / 0) and 2 min (minutes) (w1 / o / w2), respectively. After removal of the organic solvent by vaporization, precipitation of the polymer and subsequent encapsulation of the protein occurred. The particle size was determined with the help of an electron microscope. Resomer® R202H particles (FIG. 18) combined with the polyvinyl alcohol or poloxamer188 emulsifying agent gave the best results regarding particle morphology and protein loading. The average size of particles loaded with GDF-5 was approximately 320 nm, and the average drug concentration was 66 μg per mg of particles. Due to the relatively low protein load, the tested organic nanoparticles were considered to be appropriate drug carriers for cosmetic compositions comprising GDF-5 related proteins, since cosmetics contain comparatively low amounts of active ingredients.

B. Lipid microparticle compositions Certain lipid formulations are especially suitable for dermal purposes. Several compositions of lipid microparticles (LMP) were created and tested as colloidal drug carriers for cosmetic compositions comprising GDF-5 related proteins.

one.

 LMP compositions comprising natural (vegetable) natural oils For example, 22.93 µl of olive oil (21.1 mg) were combined with an equal weight of phosphatidylserine within a centrifuge microtube of 1.5 ml capacity. 956 µl of a 50 mM succinate buffer, pH 4.25, was added to bring the total volume to 1 ml. The mixture was vortexed for 30 seconds and placed in an ice and ethanol bath to keep the temperature constantly at 4 ° C. The sample was treated with ultrasound for one hour with an average power output of 22 watts in a 25% duty cycle with a cycle period of 20 seconds. After 1 hour, the emulsion appeared transparent with an amber color when held in the presence of light, indicating that the emulsion spheres were becoming smaller and had reached the expected diameter of approximately 50 nm (FIG. 19). At this point, the LMP mixture was transferred with a pipette to a centrifuge microtube containing 2.5 mg of the lyophilized GDF-5 protein. The sample was vortexed until the protein became solubilized. The introduction of the protein caused the mixture to become opaque, indicating a partial rupture of the LMP. The LMP mixture was then treated with ultrasound under the same conditions for an additional 45 minutes, restoring the previous transparency. At this point, the mixture of lipid microparticles was vortexed in a low setting while 0.5 M NaOH was added in 10 µl increments until the pH of 7.0 had been reached. The pH was measured by placing a small amount of a solution on a strip to measure the pH. In some cases, the solutions were further filtered through a 0.2 μm filter. The protein content of the lipid microparticles loaded with GDF-5 was determined through the bicinconinic acid (BCA) assay (Smith, P.K. et al. (1985), Anal. Biochem. 150, 76-85). The concentration of GDF-5 was of the expected order (about 2.5 mg / ml).

No precipitation was observed during titration indicating that the lipid microparticle composition was suitable for transporting high amounts of GDF-5 (up to 2.5 mg / ml) even at a neutral pH. As a consequence, the LMP’s tested were considered as appropriate drug vehicles for cosmetic compositions comprising GDF-5 related proteins.

2.

 LMP compositions comprising triglycerides

Composition example: Myglyol® 812 0.95 g Phospholipon® 80 0.925 g pH citrate buffer 4.25 6.7 ml GDF-5 (4.4 mg / ml) 0.227 ml NaOH 0.5 M 1 ml Water to 10 ml

Triglycerides (eg Myglyol® 812), emulsifying agents (eg Phospholipon® 80) and a citrate buffer were combined and vortexed for 30 s at 3,000 rounds per minute). The sample was treated with ultrasound for one hour with an average power output of 22 watts and continuous cooling in an ice bath. At this point, a solution of GDF-5 was added to the LMP mixture. The sample was then treated with vortex and ultrasound under the same conditions for an additional hour. The mixture of lipid microparticles was vortexed in a low setting, while 0.5 M NaOH was added in 10 µl increments until the pH of 7.0 was reached.

3. Composition of LMP without any oil phase

Composition example: Phospholipon® 80 0.925 g

PH citrate buffer 4.25 7.7 ml

GDF-5 (4.4 mg / ml) 0.227 ml

0.5 M NaOH 1 ml

Water up to 10 ml

Production instructions: see LMP compositions comprising triglycerides.

C. Microemulsions

Composition example:

Glyceryl Isoestearate 0.05 g

Phospholipon® 80

0.05 g

Isoceteth-20

0.25 g

Isopropyl Myristate

0.15 g

Glycerol

0.5 g

PH 4.25 citrate buffer

7.7 ml

GDF-5 (4.4 mg / ml)

0.227 ml

0.5 M NaOH

1 ml

Water

up to 10 ml

A w / o type microemulsion (water dispersed in an oil) was created as follows: Glyceryl isostearate, Phospholipon® 80, lsoceteth-20 and isopropyl myristate) and slowly stirred (a 300 rounds per minute) at 100 ° C to form a lipid phase. Water soluble compounds (glycerol, a citrate buffer and a growth factor solution) were combined separately. After cooling to room temperature, the water soluble mixture was added to the lipid phase in small portions. A homogeneous distribution was achieved by further agitation.

Example 5: Test of colloidal formulations of GDF-5

The biological activity of colloidal formulations of GDF-5 according to Example 4 was tested by means of classical tests with alkaline phosphatase as described eg in Example 3. According to the test results, all Colloidal formulations (polymeric nanoparticles, lipid microparticles and microemulsions) presented as characteristic a considerable bioactivity related to GDF-5. In addition, the encapsulated GDF-5 integrity test was checked by reverse phase HPLC and SDS-PAGE methods.

In order to compare the formulations of LMP with GDF-5 with a reference standard of GDF-5 and obtain information about a possible conglomeration or degradation, a common analysis by RP-HPLC (reverse phase liquid chromatography) was used. of LMP formulations of GDF-5, which separates the molecules according to their polarity. Chromatography took place in a stationary phase (column: Vydac C18, 5 μm, phase 218TP52, protein and peptide) and a mobile phase under high pressure. Elution of the protein and potential impurities during chromatography was achieved by a gradient of the mobile phase from polar (0.15% trifluoroacetic acid in water with 35% acetonitrile) to less polar (0.15% of trifluoroacetic acid in water with 100% acetonitrile). For RP-HPLC analysis the samples were centrifuged for 10 minutes at

13,000 xg. Supernatants were diluted with 0.15% TFA in 35% acetonitrile. The samples were centrifuged again and the supernatants were applied to the RP-HPLC. No GDF-5 could be detected in the supernatant material, indicating that GDF-5 had been completely immobilized and enveloped by lipids.

For the detection of potential protein degradation, microparticle granules with the encapsulated GDF-5 were heated for 10 min at 80 ° C, in order to solubilize the lipids and were further elaborated by means of an SDS-PAGE. The protein size separation (see FIG. 22) showed only very few degradation products and an integrity of most of the GDF-5 protein.

Finally, a satisfactory penetration test was performed in order to evaluate the dermal absorption of the compositions of the invention containing GDF. Various techniques for the investigation of dermal absorption and percutaneous penetration have been developed. For ethical reasons, this test should be carried out with skin samples removed by means of consecrated in vitro tests. The following classic in vitro protocol for dermal absorption and percutaneous penetration of compositions designed for single or repeated topical administration has been developed by W. Diembeck and H.-J. Duesing of Beiersdorf AG, Germany. This and other test systems that use the principles outlined in the “new proposal for OECD guidelines for in vitro percutaneous absorption of chemical agents” are also described in the Diembeck et al 1999 quotation: Test guidelines for in vitro assessment of dermal absorption and percutaneous penetration of cosmetic ingredients [in-vitro testing guidelines for dermal absorption and percutaneous penetration of cosmetic ingredients]. Food and Chemical Technology 37, 191-205.

The experiments are carried out on the unboiled dorsal skin of selected female sows (with an age of approximately 130 days and a weight of approximately 100 kg). Fresh dorsal skin (approximately 30 x 40 cm) is cut into strips of approximately 10 x 40 cm. Most of the subcutaneous fat layer is removed and the skin is rinsed with moderately hot tap water. A circular punch with an internal diameter of 5 cm is used to cut skin discs that fit inside the penetration cells. The discs are placed in plastic bags placed under vacuum and stored at -20 ° C until use. The test samples are mixed with radiologically labeled test substances and thoroughly mixed. A sufficiently marked material should provide a specific activity of approximately 0.4 Mbq / mg. Melted skin discs are rubbed dry with cotton wool.

The skin is mounted on the receiver compartment of a penetration cell (see FIG. 23) with the epidermal side facing up, and the donor chamber was held in place. The test sample is applied on the epidermal skin side and distributed evenly within the circular area of application (diameter 2.5 cm) of the penetration cell. After 24 hours, the adsorption / penetration experiment is terminated. Experiments are usually carried out in triplicate. Analytical samples are prepared as follows:

The upper penetration cell is placed within 15 ml of an appropriate solvent that is capable of solubilizing the test substance out of the emulsion and removing the residues that are adhered to the glass. A 1 ml aliquot is added to 10 ml of a scintillation cocktail for radiological analysis. After the skin disc has been removed from the lower penetration cell, approximately 5 ml of a receiving fluid is introduced by pipette into a flask. The bottom side of the skin disc and the lower penetration cell are washed in each case with 1 ml of distilled water, which is then added to the receiving fluid. The flask is filled to 10 ml with distilled water. An aliquot of 1 ml is added to 10 ml of a scintillation cocktail for radiological analysis. The excess test sample is removed from the skin surface and transferred to a scintillation vial.

The corneal layer (stratum corneum) is detached from the skin treated with an adhesive tape (Tesafilm type 4129, width 25 mm, from Beiersdorf, Germany). The strips are placed inside scintillation vials. The epidermis and dermis are separated by heat by placing the skin samples, with the epidermal side down, under light pressure on a hot plate at 80 ° C for 45 seconds. The epidermis is detached from the dermis with a spatula and solubilized in 2 ml of Soluene (from Packard). After cooling to room temperature, 10 ml of a scintillation cocktail is added to the epidermis solution. The outer hard edge of the dermis, which does not contain the test substance, is separated by cutting and discarded. The remaining dermis is cut into pieces and dissolved in 12 ml of Soluene (from Packard) for 24 hours. After cooling to room temperature, 10 ml of a scintillation cocktail is added to 1 ml of a dermis solution.

The radioactivity of all samples is measured with a liquid scintillation counter of type LSC3801 (from Beckman Instruments). The radioactivity in all samples is used to calculate the total radioactivity not absorbed (skin surface and upper cell surface) and the radioactivity absorbed (corneal layer, epidermis, dermis, receptor fluid). The sum of these values (total recovered radioactivity) was normalized to 100%. The percentages of radioactivity, on the surface, in the corneal layer, etc., were recalculated as percentages of the total normalized radioactivity to provide information about the distribution of the test substance applied (the non-absorbed versus the absorbed) . The sum of the normalized absorbed radioactivities was adjusted to 100% and the data of the corneal layer, epidermis, dermis and receiving fluid were recalculated as percentages of normalized absorbed radioactivity to provide information about the distribution of the test substance inside the skin.

Additional satisfactory permeation experiments were performed using heat-separated human skin samples from a skin donor in static Franz diffusion cells (skin surface 3 cm2). 300 μl of the topical formulations (solutions) were applied. Samples were taken at 3 times (time points) during a 48-hour study period. All tests were performed in triplicate for each formulation.

Claims (12)

one.

Cosmetic, non-therapeutic method, for the prevention, deceleration or inversion of dermal aging processes, selected from the set consisting of chronological aging and photoaging, where the composition of the extracellular matrix (ECM) of a mammal is altered by use of one or more protein (s) related to GDF-5 and / or one or more nucleic acid (s) encoding said protein (s), wherein a protein GDF-5 related is any naturally occurring or artificially created protein that comprises a cystine knot domain with an amino acid identity of at least 60% with the 102 aa cystine knot domain of GDF-5 human (amino acids 400-501 of SEQ ID NO: 1).

2.

 The method of claim 1, wherein the ECM content of a substance selected from the group consisting of type I collagen, type III collagen, type IV collagen, type VII collagen, fibrillins is increased. 1 and -2, hyaluronic acid, elastin and decorin.

3.

The method of any one of the preceding claims, said method being further characterized in that it comprises a topical, transdermal or subcutaneous administration of a composition containing use of one or more protein (s) related to GDF-5 and / or one or more nucleic acid (s) encoding said protein (s).

Four.

 A cosmetic, non-therapeutic use of a composition comprising use of one or more protein (s) related to GDF-5 and / or one or more nucleic acid (s) encoding said (s) protein (s), in which a GDF-5-related protein is any naturally occurring or artificially created protein that comprises a cystine knot domain with an amino acid identity of at least 60% with the 102 aa cystine knot domain of human GDF-5 (amino acids 400-501 of SEQ ID NO: 1) stops the improvement and / or maintenance of the dermal aspect, selected from a) improvement or prevention of wrinkles, b) delay of dermal aging, c) thickening of a basement membrane, and d) reduction of epidermal thickness.

5.

 The use according to claim 4, wherein the concentration of said GDF-5 related protein in the composition is between 0.1 μg / ml and 10 μg / ml.

6.

The use according to any one of claims 4 and 5, wherein said GDF-5 related protein is a mammalian GDF-5 protein and / or a variant thereof, wherein said variant means a biologically fragment. active of a GDF-5 protein, an artificial construction of biologically active protein, which contains additional sequences in excess of the original sequence of the GDF-5 protein or any combination thereof.

7.

 The use according to any one of claims 4 to 6, wherein said composition comprises a mature human GDF-5 or a recombinant mature human GDF-5 (amino acids 1-120 or 2-120 of SEQ ID NO: 3 ) or a recombinant monomeric human GDF-5 (amino acids 1-120 or 2-120 of SEQ ID NO: 4).

8.

 The use according to any one of claims 4 to 7, wherein the composition comprises a colloidal drug vehicle.

9.

 The use according to claim 8, wherein said composition comprises polymeric nanoparticles and / or lipid microparticles.

10.

 The use according to any one of claims 4 to 9, wherein said composition is administered topically, transdermally or subcutaneously.

eleven.

 The use according to any one of claims 4 to 10, wherein said composition is administered in a form that is selected from the set consisting of a lotion, a gel, a cream, an ointment, an atomization formulation of spray, in lipstick, a patch or plaster, a facial pack, a bath agent, a facial cleansing agent, a bath detergent, a bath soap, a sunscreen, a sun lotion, a lotion for after sun exposure, a self-tanning lotion, a microemulsion, an emulsion of the oil-in-water type, an emulsion of the water-in-oil type, an emulsion of the type w / o / w, an emulsion of the type o / w / o or a milky lotion.

12.

 The use according to any one of claims 4 to 11, wherein said composition further comprises at least one ingredient selected from the set consisting of hyaluronic acid, a retinoid, an antioxidant, an alpha-hydroxy acid, a peptide, a growth factor, a preservative agent, a fragrance or perfume, an oil, an alcohol, a fatty ester, a UV filter substance and a vitamin.